The mTOR complex 1 (mTORC1) pathway promotes cell growth in response to many cues, including amino acids, which act through the Rag guanosine triphosphatases (GTPases) to promote mTORC1 translocation to the lysosomal surface, its site of activation Although progress has been made in identifying positive regulators of the Rags, it is unknown if negative factors also exist Here, we identify GATOR as a complex that interacts with the Rags and is composed of two subcomplexes we call GATOR1 and -2 Inhibition of GATOR1 subunits (DEPDC5, Nprl2, and Nprl3) makes mTORC1 signaling resistant to amino acid deprivation In contrast, inhibition of GATOR2 subunits (Mios, WDR24, WDR59, Seh1L, and Sec13) suppresses mTORC1 signaling, and epistasis analysis shows that GATOR2 negatively regulates DEPDC5 GATOR1 has GTPase-activating protein (GAP) activity for RagA and RagB, and its components are mutated in human cancer In cancer cells with inactivating mutations in GATOR1, mTORC1 is hyperactive and insensitive to amino acid starvation, and such cells are hypersensitive to rapamycin, an mTORC1 inhibitor Thus, we identify a key negative regulator of the Rag GTPases and reveal that, like other mTORC1 regulators, Rag function can be deregulated in cancer

A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1

https://ejmcm.com/article_1158_f1a637d7625fcd2199a78af98c9bfa81.pdf

Metabolomics for the masses: The future of metabolomics in a personalized world

Synovial and plasma glucosamine concentrations in osteoarthritic patients following oral crystalline glucosamine sulphate at therapeutic dose

Integration of GC-MS and LC-MS for untargeted metabolomics profiling.

The role of liquid chromatography coupled with mass spectrometry (LC-MS) techniques in routine therapeutic drug monitoring activity is becoming increasingly important. This paper reviews LC-MS methods published in the last few years for certain classes of drugs subject to therapeutic drug monitoring: immunosuppressants, antifungal drugs, antiretroviral drugs, antidepressants and antipsychotics. For each class of compounds, we focussed on the most interesting methods and evaluated the current role of LC-MS in therapeutic drug monitoring.

http://aether.cmi.ua.ac.be/artikels/MB_32141.pdf

Current role of LC-MS in therapeutic drug monitoring.

SummaryBackground
Recent studies have shown that dysregulated metabolic pathways are linked to psoriasis pathogenesis. However, an extensive, unbiased metabolic analysis in psoriatic patients has not been completely explored. The metabolome represents the end products of proteomics or cellular processes which may be closely associated with the pathogenesis of psoriasis.

Objectives
To determine the differences in serum metabolomic profiles among psoriasis patients and healthy controls with the goal of identifying potential biomarkers in patients with psoriasis.

Methods
Serum metabolomic profiles from 29 subjects (14 psoriasis patients and 15 age and gender matched healthy controls). The serum metabolites were analyzed by gas chromatography mass spectrometry based on a combined full scan and selected-ion monitoring mode.

Results
Multivariate statistical analysis of metabolomics data revealed altered serum metabolites between the patients with psoriasis and healthy individuals. Compared to healthy individuals, psoriasis patients had higher levels of amino acids including asparagine, aspartic acid, isoleucine, phenylalanine, ornithine and proline, and higher levels of lactic acid and urea; while a lower level of crotonic acid, azelaic acid, ethanolamine and cholesterol.

Conclusions
It appears that glycolysis pathway and amino acid metabolic activity are increased in psoriasis patients. These metabolic perturbations may stem from increased demand for protein biosynthesis and keratinocyte hyperproliferation. Our findings may help elucidate the pathogenesis of psoriasis and provide insights into early diagnosis and therapeutic intervention.

This article is protected by copyright. All rights reserved.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/bjd.15008

Exploration of candidate biomarkers for human psoriasis based on gas chromatography-mass spectrometry serum metabolomics

Quantification of total and free mycophenolic acid in human plasma by liquid chromatography with fluorescence detection.

This study aimed to investigate the pharmacokinetic characteristics of total and free mycophnolic acid (MPA) and its 7-O-glucuronide metabolite (MPAG) in Chinese renal transplant recipients. In addition, limited sampling strategies were developed to estimate the individual area under concentration curve (AUC) of total and free MPA. Total and free MPA and MPAG concentrations were determined by high performance liquid chromatography. Whole 12-h pharmacokinetic profiles were obtained on the 10th day after operation in 12 adult Chinese de novo renal transplant recipients administrated with mycophenolate mofetil (MMF, 750 mg bid), cyclosporine and corticosteroids. Limited sampling strategies with jackknife technique, a resampling method, and Bland-Altman analysis were employed to develop equations to estimate total and free MPA AUC. The pattern of total and free MPA and MPAG plasma concentration-time curves in the cohort of patients taking lower doses of MMF was consistent with previous reports of Caucasian patients taking MMF 1 g bid, except that dose-normalized exposure of total and free MPAG was much lower in the current study than in those of the Caucasians. The mean C
 max and AUC0–12h of total and free MPA were 9.4 ± 3.4 mg/L, 20.2 ± 6.5 mg·h/L and 0.4 ± 0.4 mg/L, 0.7 ± 0.5 mg·h/L, respectively, whereas mean C
 max and AUC0–12h of total and free MPAG were 97.3 ± 32.6 mg/L, 656.0 ± 148.0 mg·h/L and 29.9 ± 8.5 mg/L, 222.0 ± 58.1 mg·h/L respectively. The mean fractions of free MPA and MPAG were 3.5 ± 2.0 and 34.6 ± 8.0%, respectively. No determinant was identified to influence the pharmacokinetics of total and free MPA and MPAG or the free fraction of MPA and MPAG. The combinations of C
 2h−C
 4h and C
 1h-C
 2h-C
 3h were the best to estimate free and total MPA AUC0–12h respectively, whereas the combination of C
 2h-C
 3h-C
 4h and C
 1h-C
 2h-C
 4h was the best to estimate both simultaneously. This is the first time that the pharmacokinetics profile of total and free MPA and its main metabolite MPAG has been examined in Chinese adult renal transplant patients. The limited sampling strategies proposed to estimate individual free and total MPA AUC could be useful in optimizing patient care.

Total and free mycophenolic acid and its 7-O-glucuronide metabolite in Chinese adult renal transplant patients: pharmacokinetics and application of limited sampling strategies.

A reliable and validated reversed-phase high-performance liquid chromatography (HPLC) method using fluorescence detection is reported for the simultaneous quantitation of mycophenolic acid (MPA) and valproic acid (VPA) in human plasma. The method is based on the pre-column derivatization of valproic acid with 4-bromomethyl-6, 7-dimethoxycoumarin (BrMMC) and online solvatochromism of MPA by pH adjustment. The linear calibration range was 0.50-30 microg/mL for MPA and 5.00-150 microg/mL for VPA. The relative standard deviations of the method of intra- and inter-day analyses (n = 6) were below 6.5 and 6.7% for MPA, and 5.8 and 6.3% for VPA, respectively. Dichloromethane was used for the simultaneous extraction of MPA and VPA from acidified plasma. This reliable method can be applied in the analysis of MPA and VPA in human plasma using only a small volume (100 microL).

Simultaneous determination of mycophenolic acid and valproic acid based on derivatization by high-performance liquid chromatography with fluorescence detection.

We employed CE to identify mixtures of the toxic alkaloids lappaconitine, bullatine A, atropine sulfate, atropine methobromide, scopolamine hydrobromide, anisodamine hydrobromide, brucine, strychnine, quinine sulfate, and chloroquine in human blood and urine, using procaine hydrochloride as an internal standard. The separation employed a fused-silica capillary of 75 microm id x 60 cm length (effective length: 50.2 cm) and a buffer containing 100 mM phosphate and 5% ACN (pH 4.0). The sample was injected in a pressure mode and the separation was performed at a voltage of 16 kV and a temperature of 25 degrees C. The compounds were detected by UV absorbance at wavelengths of 195 and 235 nm. All the ten alkaloids were separated within 16 min. The method was validated with regard to precision (RSD), accuracy, sensitivity, linear range, LOD, and LOQ. In blood and urine samples, the detection limits were 5-40 ng/mL and linear calibration curves were obtained over the range of 0.02-10 microg/mL. The precision of intra- and interday measurements was less than 15%. Electrophoretic peaks could be identified either by the relative migration time or by their UV spectrum.

Yunqiu Yu

Papers

Exploration of candidate biomarkers for human psoriasis based on gas chromatography-mass spectrometry serum metabolomics

Quantification of total and free mycophenolic acid in human plasma by liquid chromatography with fluorescence detection.

Total and free mycophenolic acid and its 7-O-glucuronide metabolite in Chinese adult renal transplant patients: pharmacokinetics and application of limited sampling strategies.

Simultaneous determination of mycophenolic acid and valproic acid based on derivatization by high-performance liquid chromatography with fluorescence detection.

Application of high performance capillary electrophoresis on toxic alkaloids analysis.